Chemical Constituents from the Aerial Parts of Artemisia iwayomogi and Their Anti-Neuroinflammatory Activities

Neuroinflammation, predominantly mediated by microglial activation, is a key immunological response in the pathogenesis of neurodegenerative disorders. In our preliminary study, the aerial part of Artemisia iwayomogi inhibits LPS-induced microglial activation. The present study aims to identify chemical constituents with anti-neuroinflammatory properties in the aerial parts of A. iwayomogi. Two new guaianolide sesquiterpenes, iwayomogins A and B (1 and 2), along with thirteen known sesquiterpene lactones (3–15), one diterpene glycoside (16), and nine phenolic compounds (17–25) were isolated from the aerial parts of A. iwayomogi by repeated chromatography. The structures of the isolates were elucidated by their spectroscopic data. All isolates were evaluated for their inhibitory activities on nitric oxide (NO) production in LPS-induced BV-2 microglial cells. 2,3-Dehydro-1-epi-asperilin (11) exhibited the strongest inhibitory effect on NO production (IC50 value of 1.78 μM). In the molecular docking study, three compounds (1, 2, and 11) showed good binding affinities with iNOS. Additionally, compounds 1, 2, and 11 inhibit pro-inflammatory cytokines (TNF-α and IL-6) in dose-dependent manners. The present study demonstrates that the chemical constituents from A. iwayomogi inhibit NO production and pro-inflammatory cytokine release in BV-2 cells. However, further evaluation with biological experiments utilizing in vivo models is necessary.


Introduction
Neuroinflammation is an inflammatory response within the central nervous systems (CNS) which can be activated by a variety of neuronal insults, such as infection, trauma, and toxins [1]. Microglia, as the resident of macrophages in the CNS, induce a systematic neuroinflammation process that can cause neurodegenerative diseases including Alzheimer's disease, Parkinson's disease, multiple sclerosis, and prion disease [2]. When microglia are activated by various stimuli, including lipopolysaccharides (LPS), pro-inflammatory mediators are released, which can lead to neuronal death and neurogenesis inhibition [3]. For these reasons, to prevent neurodegenerative disorders, it is necessary to investigate the drugs that suppress the inflammatory mediators in microglia [4].
In our preliminary study, the EtOH extract from the aerial parts of A. iwayomogi significantly inhibited neuroinflammation induced by LPS in the murine microglial BV-2 cells of mice by suppressing pro-inflammatory mediators and NF-κB and MAPK pathways [15]. However, active compounds related to inflammation mediated CNS disorders have still not been detected in A. iwayomogi. Therefore, the present study aims to identify chemical constituents with anti-neuroinflammatory properties in the aerial parts of A. iwayomogi. In the present study, we conducted an investigation into novel bioactive compounds in the 90% EtOH extract of A. iwayomogi by repetitive chromatographic purification. To explore the inhibitory effects of the isolates on neuro-inflammation, we treated the compounds with BV-2 microglial cells and assessed their inhibitory activities on inflammatory mediators. We further investigated the interactions between iNOS with active compounds using molecular docking studies to determine whether they could be used in the treatment of NO-mediated inflammatory diseases.

Structure Elucidation of the Compounds Isolated from A. iwayomogi
Repeated chromatography led to the isolation of two new guaianolide sesquiterpenes, iwayomogins A and B (1 and 2), along with ten sesquiterpene lactones (3)(4)(5)(6)(7)(8)(9)(10)(11)(12), three sesquiterpenes (13)(14)(15), one diterpene glycoside (16), two coumarins (17 and 18), three flavonoids (19)(20)(21), and four phenolic compounds (22)(23)(24)(25) from the aerial parts of A. iwayomogi ( Figure 1). iwayomogi has anti-inflammatory, anti-oxidative, anti-allergic, anti-obesity, and a CCl4-induced liver fibrosis inhibitory properties [9][10][11][12][13]. Notably, seco-guaianolide-type sesquiterpenes and coumarins isolated from A. iwayomogi inhibited inducible NOS (iNOS) expression in LPS-activated macrophages [14]. In our preliminary study, the EtOH extract from the aerial parts of A. iwayomogi significantly inhibited neuroinflammation induced by LPS in the murine microglial BV-2 cells of mice by suppressing pro-inflammatory mediators and NF-κB and MAPK pathways [15]. However, active compounds related to inflammation mediated CNS disorders have still not been detected in A. iwayomogi. Therefore, the present study aims to identify chemical constituents with anti-neuroinflammatory properties in the aerial parts of A. iwayomogi. In the present study, we conducted an investigation into novel bioactive compounds in the 90% EtOH extract of A. iwayomogi by repetitive chromatographic purification. To explore the inhibitory effects of the isolates on neuro-inflammation, we treated the compounds with BV-2 microglial cells and assessed their inhibitory activities on inflammatory mediators. We further investigated the interactions between iNOS with active compounds using molecular docking studies to determine whether they could be used in the treatment of NO-mediated inflammatory diseases.

Inhibitory Effects of the Isolates on NO Production
Nitric oxide (NO) is one of the crucial signaling molecules that mediates the inflammatory processes [35]. In order to determine which isolates from A. iwayomogi exert regulatory activities on NO production, all isolates were treated with LPS on BV-2 microglial cells and the concentration of NO in the supernatant of the treated cells was measured. 10-Desmethyl-1-methyl-5,6-dihydroeudesma-1,3,5(10)-triene-12,8β-olide (10) and lumiyomogin (12) were excluded from the comparison because of their considerable cytotoxicity at 10 µM concentration, although none of the other compounds showed significant cytotoxicity.

Molecular Docking Studies of the Active Compounds
NO is synthesized by three types of nitric oxide synthase with L-arginine and cofactors, and iNOS plays an important role in the NO production process [36]. Therefore, selective iNOS inhibitory agents have been studied by several researchers [37]. To further investigate the effects of the compounds reducing NO concentration on the inhibition of iNOS protein (PDB code: 3E6T), the most active compound (11) and the new compounds (1 and 2) were selected for an in silico molecular docking study (Figure 4). Compounds 11, 1, and 2 showed strong binding affinities (−9.0, −8.0, and −8.0 kcal/mol, respectively) with iNOS. At the lowest energy binding mode, compound 11 interacted with both ALA-345 and HEM-901, which are involved in hydrogen bonding. In addition, compound 1 interacted with GLN-257 via hydrogen bonding, although the binding affinities were the same in the lowest docking conformation for compounds 1 and 2. The data also suggests that the active compounds may reduce NO production in BV-2 microglial cells by inhibiting iNOS activity. tors, and iNOS plays an important role in the NO production process [36]. Therefore, selective iNOS inhibitory agents have been studied by several researchers [37]. To further investigate the effects of the compounds reducing NO concentration on the inhibition of iNOS protein (PDB code: 3E6T), the most active compound (11) and the new compounds (1 and 2) were selected for an in silico molecular docking study (Figure 4). Compounds 11, 1, and 2 showed strong binding affinities (−9.0, −8.0, and −8.0 kcal/mol, respectively) with iNOS. At the lowest energy binding mode, compound 11 interacted with both ALA-345 and HEM-901, which are involved in hydrogen bonding. In addition, compound 1 interacted with GLN-257 via hydrogen bonding, although the binding affinities were the same in the lowest docking conformation for compounds 1 and 2. The data also suggests that the active compounds may reduce NO production in BV-2 microglial cells by inhibiting iNOS activity.

Inhibitory Effects on Pro-Inflammatory Cytokines
TNF-α and IL-6 are typical pro-inflammatory cytokines, which contribute to inflammatory propagation and aggravation [38,39]. To investigate the molecular mechanism of the most potent compound, 2,3-dehydro-1-epi-asperilin (11), and the new compoundsiwayomogins A and B (1 and 2)-on neuroinflammatory response, the concentrations of the pro-inflammatory cytokines in the supernatant of the treated cells were measured. The results showed that TNF-α and IL-6 levels were markedly elevated in the LPS-only-treated group compared with the control group. However, compounds 1, 2, and 11 reduced cytokine concentrations in dose-dependent manners ( Figure 5). These results indicate that compounds 1, 2, and 11 are effective to control neuroinflammation via simultaneous regulation of pro-inflammatory mediators. These effects are thought to be related to interactions with iNOS and the consequent regulation of NO production, resulting in the cessation of the overall inflammatory process, including the release of cytotoxic cytokines. Considering that neuroinflammation is one of the contributors in various psychological disorders and neurodegenerative diseases, compounds 1, 2, and 11 are anticipated to be potential candidates for treatment of neurological disorders due to their anti-neuroinflammatory effects.
cate that compounds 1, 2, and 11 are effective to control neuroinflammation via simultaneous regulation of pro-inflammatory mediators. These effects are thought to be related to interactions with iNOS and the consequent regulation of NO production, resulting in the cessation of the overall inflammatory process, including the release of cytotoxic cytokines. Considering that neuroinflammation is one of the contributors in various psychological disorders and neurodegenerative diseases, compounds 1, 2, and 11 are anticipated to be potential candidates for treatment of neurological disorders due to their anti-neuroinflammatory effects. Values are indicated as the mean ± SEM. Data were analyzed by one-way ANOVA, followed by Tukey's post-hoc test. ### p < 0.001 compared with the control group; * p < 0.05, ** p < 0.01, and *** p < 0.001 compared with the LPS-only-treated group.  IL-6 (D-F) were measured using ELISA kits. Control group, vehicle-only-treated group (white bar); LPS-treated group (black bar); LPS with compounds-treated groups (gray bars). Values are indicated as the mean ± SEM. Data were analyzed by one-way ANOVA, followed by Tukey's post-hoc test. ### p < 0.001 compared with the control group; * p < 0.05, ** p < 0.01, and *** p < 0.001 compared with the LPS-only-treated group. plates. MPLC (Teledyne Isco, Lincoln, NE, USA) was used for further fractionation with prepacked Redi Sep-Silica (12 g, 24 g, 40 g, Teledyne Isco) and Redi Sep-C18 cartridges (26 g, 43 g, 80 g, Teledyne Isco). Preparative HPLC was performed using a HPLC purification system (1525 pump and PDA 1996 detector, Waters Corp., Milford, MA, USA) with a Gemini NX-C18 110A (250 × 21.2 mm i.d., 5 µm, Phenomenex, Torrance, CA, USA) or YMC-pack ODS-A columns (250 × 20.0 mm i.d., 5 µm, YMC Co., Ltd., Kyoto, Japan). UV and FT-IR spectra were recorded using OPTIZEN UV-Vis and Agilent Cary 630 FT-IR (Agilent Technologies, Santa Clara, CA, USA) spectrophotometers, respectively. Optical rotations were measured using a JASCO P-2000 polarimeter; NMR spectra were acquired by JEOL (JEOL, Tokyo, Japan) at 500 MHz; and HR-DART-MS spectra were obtained by the DART ion source (Ionsense, Tokyo, Japan) coupled to an AccuTOF-TLC (JEOL). Finally, the circular dichroism spectra were measured by a Chirascan-plus spectrometer (Applied Photophysics Ltd., Leatherhead, UK).

Plant Material
The aerial parts of Artemisia iwayomogi Kitamura (Compositae) were purchased from Kwangmyoungdang Pharmaceutical Co., Ltd. (Ulsan-si, Korea) in January 2019. The origin of the herbal material was identified by Professor Myeong Sook Oh, and a voucher specimen (ARIW-2019) was deposited in the Laboratory of Natural Product Medicine, College of Pharmacy, Kyung Hee University (Seoul, Korea).

Extraction and Isolation
The dried plant materials (3.0 kg) were extracted twice with 90% EtOH (30 L) over the course of 48 h at room temperature, then the solvent was removed in vacuo at 45 • C to give a 90% EtOH extract (300 g). The extract was then suspended in distilled water (0.6 L) and partitioned three times with EtOAc (0.6 L). The EtOAc-soluble fraction (130 g) was subjected to Diaion HP-20 CC (11.3 × 55.6 cm) using an acetone-water gradient system (from 30:70 to 100:0, v/v) to obtain 19 fractions (F1-F19). Compound 18 (1.59 g) was purified by recrystallization from F3 in cold acetone. After recrystallization, the supernatant of F3 (18.  Table 1).  Table 1).

Computational Methods for the ECD Spectrum
The 3D models of active compounds were built using Chem3D, and the random conformational analysis was performed with the Merk molecular force field (MMFF) implemented by the Spartan'14 software program (Wavefunction, Inc., Irvine, CA, USA; 2014). Geometrical optimization of the selected lowest energy conformers was performed at the B3YLP/6-31+g(d, p) level using Gaussian 09 (Revision E.01; Gaussian, Inc., Wallingford, CT, USA; 2009). The electronic circular dichroism (ECD) calculations of the optimized conformers were calculated using the time-dependent density functional theory (TDDFT) method at the CAM-B3LYP/SVP level with the conductor-like polarizable continuum solvent model (CPCM, methanol). The final ECD spectra were generated by Boltzmann weighting each conformer.

Cell Culture and Treatment
BV-2 microglial cells were maintained in Dulbecco's Modified Eagle's medium (Hyclone Laboratories, Inc., Logan, UT, USA), supplemented with 10% fetal bovine serum (Hyclone Laboratories) and 1% penicillin-streptomycin (Hyclone Laboratories), and incubated at 37 • C in a humidified atmosphere containing 5% CO 2 . All experiments were carried out 24 h after seeding in 96-well or 12-well plates at a density of 3.0 × 10 5 cells/mL. The following day, the cells were pre-treated with various concentrations of the compounds in serum-free media for 1 h, before being stimulated with 100 ng/mL of LPS (Sigma-Aldrich, St. Louis, MO, USA) for an additional 23 h. An equal volume of vehicle was given to the control and each of the toxin groups.

Measurement of Pro-Inflammatory Mediators
NO concentration was determined using the method described in the previous study [15]. The supernatant of the cells seeded in a 96-well plate was harvested and mixed with an equal volume of Griess reagent (1% sulfanilamide, 0.1% naphthylethylenediamine dihydrochloride, 2% phosphoric acid). After 10 min, the absorbance at 540 nm was measured using a microplate reader (Versamax™, Molecular Devices, LLC., San Jose, CA, USA). Sodium nitrite was used as a standard to calculate the NO concentration. TNF-α (BD Biosciences, Franklin Lakes, NJ, USA) and IL-6 (R&D Systems, Minneapolis, MN, USA) protein concentrations in the supernatant of cells seeded in the 12-well plates were assessed using enzyme-linked immunosorbent assay (ELISA) kits following the manufacturer's protocols.

Molecular Docking
The crystal structures of iNOS were obtained from the RCSB PDB database (PDB code: 3E6T, resolution: 2.5 Å). The protein was prepared by removing all water molecules and adding polar hydrogen atoms using AutoDock 4.2 software (The Scripps Research Institute, La Jolla, CA, USA). The grid box size was 30Ǻ ×30Ǻ ×30Ǻ with 0.175Ǻ. The absolute configurations of compounds 1 and 2 were confirmed by NOESY and ECD calculation data, and the 3D structures of the ligands were minimized using Chem3D Pro 14.0. After the preparation of the protein and ligands, molecular docking calculations were performed using AutoDock Vina software with AutoDock Tools 1.5.6. (The Scripps Research Institute, La Jolla, CA, USA) and using the hybrid Lamarckian Genetic Algorithm (LGA). The 2D and 3D diagrams of the protein-ligand complexes and the 2D diagrams of protein-ligand interactions were generated with Maestro 12.9 software (Schrödinger, LLC., New York, NY, USA).

Statistical Analysis
Statistical parameters were calculated using GraphPad Prism 8.0 software (GraphPad Software, San Diego, CA, USA). Values were expressed as the mean ± standard error of the mean (SEM) and analyzed using a one-way analysis of variance (ANOVA) followed by Tukey's post-hoc test. Differences with p-values of less than 0.05 were considered statistically significant.

Conclusions
The aerial parts of A. iwayomogi have been widely used in traditional Korean medicine to treat various inflammation-mediated diseases. Our findings revealed that the chemical constituents from A. iwayomogi can inhibit neuroinflammation in LPS-induced BV-2 cells by suppressing the NO production and the release of pro-inflammatory cytokines. Considering that neuroinflammation contributes to various psychological disorders and neurodegenerative diseases, these isolates could be used in the treatment of neurological disorders because of their anti-neuroinflammatory properties. Therefore, further research is necessary to confirm whether their anti-inflammatory effects would be effective against neurological disorders in in vivo models.